1. Field of the Invention
This invention relates to a method for detecting an amphiphilic antigen in a biological sample suspected of containing the amphiphilic antigen. The invention has particular application to detecting the presence of gram-negative bacteria in a clinical specimen.
Numerous techniques are known for detecting the presence of antigens in a sample, such as a biological fluid, i.e., blood, urine, cell cultures. Many of the these techniques involve cell culture procedures, which are relatively long and complicated, and which give results that are greatly dependent on the skill of the technician. Other techniques, such as electrophoresis, require complicated and/or costly instruments that are not readily available.
Many of the detection techniques involve immunoassays. Some of these techniques involve detecting antibodies to the antigen of interest. Such indirect techniques tend to be inaccurate because antibodies often remain in the human body after the disease has been cured. Therefore, it is preferable to assay for antigens rather than antibodies. Immunoassay techniques for the detection of the presence or amount of antigen in a biological fluid often involve enzyme immunoassays such as the enzyme linked immunosorbent assay, generally referred to as ELISA. Such assays typically involve detecting the antigen of interest by coating the antigen on a bare solid surface or on a surface that has been pre-coated, for example, with a protein which is usually an antibody to the antigen. The surface is then washed to remove unbound antigen. Thereafter, the antigen on the surface is contacted with an antibody for the antigen that is labeled or is capable of being labeled. The surface is again washed and the antibody that has become bound to the surface of the support is detected by detecting the label.
The number of steps required in the above procedure adds significant time and manipulation to the assay. There is, therefore, a need for a method for detecting antigens, especially antigens from gram-negative bacteria, that is more rapid, reliable and cost effective than tests now known or available. It is likewise important that such a test require less manipulation than presently available tests.
2. Related Disclosures
European Published Patent Application No. 0 303 515 discloses a method for assaying an amphipathic analyte, such as a lipoprotein or a lipopolysaccharide, wherein the lipophilic ends of the analytes are bound to a substantially homogeneous lipophilic surface of a solid substrate. Presence of the bound analyte is then determined by conventional methods.
A novel process for removing endotoxin from biological fluids and for removing or reducing the level of endotoxin from the blood of animals is disclosed in U.S. Pat. No. 3,959,128. The process disclosed involves the use of certain non-ionogenic hydrophobic synthetic plastic polymers that are capable of adsorbing endotoxin from biological fluids when placed in intimate contact therewith.
An in vitro process for detecting the presence of endotoxin in a biological fluid wherein the amebocyte lysate from the hemolymph of the horseshoe crab, Limulus polyphemus, is contacted with and incubated in the presence of a synthetic plastic polymer capable of adsorbing endotoxin which has previously been contacted with the biological fluid is disclosed in U.S. Pat. No. 3,944,391.
A method for determining Chlamydia trachomatis antigen in a clinical specimen is disclosed in U.S. Pat. No. 4,497,899. The method disclosed involves lysing Chlamydia cells in the specimen to release the antigen; coating a bare solid support with the cell lysate; separating the coated support from the specimen; treating the separated support with antibody to form an antigen-chlamydia antibody complex on the support; separating the complex from unbound antibody; treating the bound complex with labeled antiglobulin to form an antigen-antibody-labeled antiglobulin complex on the support; separating the latter complex from unbound labeled antiglobulin; and determining bound labeled antiglobulin as a measure of antigen in the specimen. U.S. Pat. No. 4,497,900 discloses a method for determining Neisseria gonorrhoeae analogous to that in U.S. Pat. No. 4,497,899.
Endotoxin-polymyxin B complexes are disclosed in Vox Sang. 1987, Vol. 52, pages 272-280, as being useful in an improved ELISA for IgG antibodies to gram-negative endotoxin core glycolipids in sera from blood donors.